Gossypol co-crystals and the use thereof

ABSTRACT

This invention relates to compositions comprising co-crystals of (−)-gossypol with a C 1-8  carboxylic acid or C 1-8  sulfonic acid which are useful as inhibitors of Bcl-2 family proteins. The invention also relates to the use of co-crystals of (−)-gossypol with a C 1-8  carboxylic acid or C 1-8  sulfonic acid for inducing apoptosis in cells and for sensitizing cells to the induction of apoptotic cell death.

This application claims priority to U.S. Provisional Application60/556,249 filed Mar. 25, 2004, and herein incorporated by reference inits entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the field of medicinal chemistry. In particular,the invention relates to compositions comprising co-crystals of(−)-gossypol with a C₁₋₈ carboxylic acid or C₁₋₈ sulfonic acid which areuseful as inhibitors of Bcl-2 family proteins. The invention alsorelates to the use of co-crystals of (−)-gossypol with a C₁₋₈ carboxylicacid or C₁₋₈ sulfonic acid for inducing apoptosis in cells and forsensitizing cells to the induction of apoptotic cell death.

2. Related Art

The aggressive cancer cell phenotype is the result of a variety ofgenetic and epigenetic alterations leading to deregulation ofintracellular signaling pathways (Ponder, Nature 411:336 (2001)). Thecommonality for all cancer cells, however, is their failure to executean apoptotic program, and lack of appropriate apoptosis due to defectsin the normal apoptosis machinery is a hallmark of cancer (Lowe et al.,Carcinogenesis 21:485 (2000)). Most of the current cancer therapies,including chemotherapeutic agents, radiation, and immunotherapy, work byindirectly inducing apoptosis in cancer cells. The inability of cancercells to execute an apoptotic program due to defects in the normalapoptotic machinery is thus often associated with an increase inresistance to chemotherapy, radiation, or immunotherapy-inducedapoptosis. Primary or acquired resistance of human cancer of differentorigins to current treatment protocols due to apoptosis defects is amajor problem in current cancer therapy (Lowe et al., Carcinogenesis21:485 (2000); Nicholson, Nature 407:810 (2000)). Accordingly, currentand future efforts towards designing and developing new moleculartarget-specific anticancer therapies to improve survival and quality oflife of cancer patients must include strategies that specifically targetcancer cell resistance to apoptosis. In this regard, targeting crucialnegative regulators that play a central role in directly inhibitingapoptosis in cancer cells represents a highly promising therapeuticstrategy for new anticancer drug design.

Two classes of central negative regulators of apoptosis have beenidentified. The first class of regulators is the inhibitor of apoptosisproteins (IAPs) (Deveraux et al., Genes Dev. 13:239 (1999); Salvesen etal., Nat. Rev. Mol. Cell. Biol. 3:401 (2002)). IAP proteins potentlysuppress apoptosis induced by a large variety of apoptotic stimuli,including chemotherapeutic agents, radiation, and immunotherapy incancer cells.

The second class of central negative regulators of apoptosis is theBcl-2 family of proteins (Adams et al., Science 281:1322 (1998); Reed,Adv. Pharmacol. 41:501 (1997); Reed et al., J. Cell. Biochem. 60:23(1996)). Bcl-2 is the founding member of the family and was firstisolated as the product of an oncogene. The Bcl-2 family now includesboth anti-apoptotic molecules such as Bcl-2 and Bcl-X_(L) andpro-apoptotic molecules such as Bax, Bak, Bid, and Bad. Bcl-2 and BclX_(L) are overexpressed in many types of human cancer (e.g., breast,prostate, colorectal, lung, etc.), including Non-Hodgkin's lymphoma,which is caused by a chromosomal translocation (t14, 18) that leads tooverexpression of Bcl-2. This suggests that many cancer cell typesdepend on the elevated levels of Bcl-2 and/or Bcl-X_(L) to survive theother cellular derangements that simultaneously both define them ascancerous or pre-cancerous cells and cause them to attempt to executethe apoptosis pathway. Also, increased expression of Bcl-2 familyproteins has been recognized as a basis for the development ofresistance to cancer therapeutic drugs and radiation that act in variousways to induce cell death in tumor cells.

Bcl-2 and Bcl-X_(L) are thought to play a role in tumor cell migrationand invasion, and therefore, metastasis. Amberger et al., Cancer Res.58:149 (1998); Wick et al., FEBS Lett, 440:419 (1998); Mohanam et al.,Cancer Res. 53:4143 (1993); Pedersen et al., Cancer Res., 53:5158(1993). Bcl-2 family proteins appear to provide tumor cells with amechanism for surviving in new and non-permissive environments (e.g.,metastatic sites), and contribute to the organospecific pattern ofclinical metastatic cancer spread. Rubio, Lab Invest. 81:725 (2001);Fernández et al., Cell Death Differ. 7:350 (2000)). Anti-apoptoticproteins such as Bcl-2 and/or Bcl-X_(L) are also thought to regulatecell-cell interactions, for example through regulation of cell surfaceintegrins. Reed, Nature 387:773 (1997); Frisch et al., Curr. Opin. CellBiol. 9:701 (1997); Del Bufalo et al., FASEB J. 11:947 (1997).

Therapeutic strategies for targeting Bcl-2 and Bcl-X_(L) in cancer torestore cancer cell sensitivity and overcome resistance of cancer cellsto apoptosis have been extensively reviewed (Adams et al., Science281:1322 (1998); Reed, Adv. Pharmacol. 41:501 (1997); Reed et al., J.Cell. Biochem. 60:23 (1996)). Currently, Bcl-2 antisense therapy is inseveral Phase III clinical trials for the treatment of solid andnon-solid tumors.

Gossypol is a naturally occurring double biphenolic compound derivedfrom crude cotton seed oil (Gossypium sp.). Human trials of gossypol asa male contraceptive have demonstrated the safety of long termadministration of these compounds (Wu, Drugs 38:333 (1989)). Gossypolhas more recently been shown to have some anti-proliferative effects(Flack et al., J. Clin. Endocrinol. Metab. 76:1019 (1993); Bushunow etal., J. Neuro-Oncol. 43:79, (1999); Van Poznak et al., Breast CancerRes. Treat. 66:239 (2001)). (−)-Gossypol and its derivatives recentlyhave been shown to be potent inhibitors of Bcl-2 and Bcl-X_(L) and tohave strong anti-cancer activity (U.S. patent application No.2003/0008924).

A composition comprising racemic gossypol and acetic acid is known inthe art (Sigma-Aldrich Corp., St. Louis, Mo.). Previous attempts tocrystallize (−)-gossypol have resulted in crystals that are too poor forX-ray analysis (Gdaniec et al., “Gossypol,” in ComprehensiveSupramolecular Chemistry (Atwood et al. eds.), Vol. 6, Pergamon) or inco-crystals of (−)-gossypol and acetone when using a solution of racemicgossypol acetic acid in acetone (Dowd et al., J. Am. Oil Chem. Soc.76:1343 (1999)).

SUMMARY OF THE INVENTION

The present invention relates to compositions comprising co-crystals of(−)-gossypol (formula I) with a C₁₋₈ carboxylic acid or C₁₋₈ sulfonicacid (“(−)-gossypol co-crystals”). These compositions are useful forinhibiting the activity of anti-apoptotic Bcl-2 family proteins,inducing apoptosis in cells, and increasing the sensitivity of cells toinducers of apoptosis.

It is generally accepted that the inability of cancer cells or theirsupporting cells to undergo apoptosis in response to genetic lesions orexposure to inducers of apoptosis (such as anticancer agents andradiation) is a major factor in the onset and progression of cancer. Theinduction of apoptosis in cancer cells or their supporting cells (e.g.,neovascular cells in the tumor vasculature) is thought to be a universalmechanism of action for virtually all of the effective cancertherapeutic drugs or radiation therapies on the market or in practicetoday. One reason for the inability of a cell to undergo apoptosis isincreased expression and accumulation of anti-apoptotic Bcl-2 familyproteins.

The present invention contemplates that exposure of animals sufferingfrom cancer to therapeutically effective amounts of (−)-gossypolco-crystal that inhibit the function(s) of anti-apoptotic Bcl-2 familyproteins will kill cancer cells or supporting cells outright (thosecells whose continued survival is dependent on the overactivity of Bcl-2family proteins) and/or render such cells as a population moresusceptible to the cell death-inducing activity of cancer therapeuticdrugs or radiation therapies. The present invention contemplates that(−)-gossypol co-crystals will satisfy an unmet need for the treatment ofmultiple cancer types, either when administered as monotherapy to induceapoptosis in cancer cells dependent on anti-apoptotic Bcl-2 familyproteins function, or when administered in a temporal relationship withother cell death-inducing cancer therapeutic drugs or radiationtherapies so as to render a greater proportion of the cancer cells orsupportive cells susceptible to executing the apoptosis program comparedto the corresponding proportion of cells in an animal treated only withthe cancer therapeutic drug or radiation therapy alone.

In certain embodiments of the invention, it is expected that combinationtreatment of animals with a therapeutically effective amount of acomposition of the present invention and a course of an anticancer agentor radiation will produce a greater tumor response and clinical benefitin such animals compared to those treated with the composition oranticancer drugs/radiation alone. Put another way, because thecompositions lower the apoptotic threshold of all cells that expressanti-apoptotic Bcl-2 family proteins, the proportion of cells thatsuccessfully execute the apoptosis program in response to the apoptosisinducing activity of anticancer drugs/radiation will be increased.Alternatively, the compositions of the present invention are expected toallow administration of a lower, and therefore less toxic and moretolerable, dose of an anticancer agent and/or radiation to produce thesame tumor response/clinical benefit as the conventional dose of theanticancer agent/radiation alone. Since the doses for all approvedanticancer drugs and radiation treatments are known, the presentinvention contemplates combination therapies with various combinationsof known drugs/treatments with the present compositions. Also, since thecompositions of the present invention act at least in part by inhibitinganti-apoptotic Bcl-2 family proteins, the exposure of cancer cells andsupporting cells to therapeutically effective amounts of thecompositions can be temporally linked to coincide with the attempts ofcells to execute the apoptosis program in response to the anticanceragent or radiation therapy. Thus, in some embodiments, administering thecompositions of the present invention in connection with certaintemporal relationships, will provide especially efficacious therapeuticpractices.

(−)-Gossypol co-crystal is useful for the treatment, amelioration, orprevention of disorders responsive to induction of apoptotic cell death,e.g., disorders characterized by dysregulation of apoptosis, includinghyperproliferative diseases such as cancer. In certain embodiments,(−)-gossypol co-crystal can be used to treat, ameliorate, or preventcancer that is characterized by resistance to cancer therapies (e.g.,those which are chemoresistant, radiation resistant, hormone resistant,and the like). In additional embodiments, (−)-gossypol co-crystal can beused to treat, ameliorate, or prevent metastatic cancer. In otherembodiments, (−)-gossypol co-crystal can be used to treathyperproliferative diseases characterized by overexpression ofanti-apoptotic Bcl-2 family proteins.

The present invention provides methods of treating a viral, microbial,or parasitic infection in an animal, comprising administering to saidanimal a therapeutically effective amount of (−)-gossypol co-crystal.

The present invention provides pharmaceutical compositions comprising(−)-gossypol co-crystal and a pharmaceutically acceptable carrier.

The invention further provides methods of making a pharmaceuticalcomposition comprising admixing (−)-gossypol co-crystal in atherapeutically effective amount to induce apoptosis in cells or tosensitize cells to inducers of apoptosis with a pharmaceuticallyacceptable carrier

The invention further provides kits comprising (−)-gossypol co-crystaland instructions for administering the composition to an animal. Thekits may optionally contain other therapeutic agents, e.g., anticanceragents.

The invention also provides methods of making (−)-gossypol co-crystal.For example, co-crystals may be prepared by a method comprisingdissolving (−)-gossypol in acetone to form a solution, filtering thesolution, adding a C₁₋₈ carboxylic acid or C₁₋₈ sulfonic acid into thesolution with mixing until the solution turns turbid, leaving the turbidsolution at room temperature then at a reduced temperature to formco-crystals, collecting the co-crystals, washing the co-crystals with asolvent, and drying the co-crystals.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows the ¹H NMR spectrum of (−)-gossypol acetic acid co-crystal.

FIG. 2 shows the ¹³C NMR spectrum of (−)-gossypol acetic acidco-crystal.

FIG. 3 shows the infrared spectrum of (−)-gossypol acetic acidco-crystal.

FIG. 4 shows the mass spectrum of (−)-gossypol acetic acid co-crystal.

FIG. 5 shows the X-ray powder diffraction spectrum of (−)-gossypolacetic acid co-crystal.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions comprising co-crystals of(−)-gossypol with a C₁₋₈ carboxylic acid or C₁₋₈ sulfonic acid(“(−)-gossypol co-crystals”), which are useful as inhibitors ofanti-apoptotic Bcl-2 family proteins. By inhibiting anti-apoptotic Bcl-2family proteins, the (−)-gossypol sensitizes cells to inducers ofapoptosis and, in some instances, itself induces apoptosis. Therefore,the invention relates to methods of sensitizing cells to inducers ofapoptosis and to methods of inducing apoptosis in cells, comprisingadministering (−)-gossypol co-crystal alone or in combination with aninducer of apoptosis. The invention further relates to methods oftreating, ameliorating, or preventing disorders in an animal that areresponsive to induction of apoptosis comprising administering to theanimal (−)-gossypol co-crystal and an inducer of apoptosis. Suchdisorders include those characterized by a dysregulation of apoptosisand those characterized by overexpression of anti-apoptotic Bcl-2 familyproteins.

The terms “(−)-gossypol,” or “(−)-gossypol compound/composition,” asused herein, refer to an optically active composition of gossypolwherein the active molecules comprising the composition rotate planepolarized light counterclockwise (e.g., levorotatory molecules) asmeasured by a polarimeter. Preferably, the (−)-gossypol compound has anenantiomeric excess of 1% to 100%. In one embodiment, the (−)-gossypolcompound has an enantiomeric excess of at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%(−)-gossypol. In one example of a “(−)-gossypol compound”, the specificrotation ([α]_(D)) of the compound is about −350° to about −390°, about−375° to about −390°, or about −385° to about −390°. (See e.g., Dowd,Chirality, 15:486 (2003); Ciesielska et al., Chem. Phys. Lett. 353:69(2992); Freedman et al., Chirality, 15:196 (2003); and Zhou et al.,Kexue Tongbao, 28:1574 (1983)). Methods for resolving racemic gossypolcompounds into substantially purified (+)- or (−)-gossypol are known(See e.g., Zhou et al., Kexue Tongbao, 28:1574 (1983) (wherein:L-phenylalanine methyl ester was mixed with the aldehyde groups ofgossypol to form a Schiff's base with two diastereoisomers which werethen resolved on a normal silica flash chromatography column. Thefiltrate was concentrated, and the residue was purified bychromatography on silica gel eluting with hexanes:EtOAc=3:1 to give twofractions. Acid hydrolysis of the two fractions in 5N HCl:THF (1:5, roomtemperature, overnight) regenerated the individual gossypol enantiomers,respectively. The first fraction with a higher R_(f) value contained(−)-gossypol, and the second fraction with a lower R_(f) value contained(+)-gossypol. The crude gossypol fractions were extracted into etherfrom the residue after removing THF from the reaction mixture. Thegossypol fractions were then purified by chromatography on silica geland eluted with hexanes:EtOAc (3:1 ratio) to give optically puregossypol, with a yield of 30-40% in two steps. The optical rotatorydispersion values for these products were α_(D)=−352° (c=0.65, CHCl₃)for (−)-gossypol, and α_(D)=+341° (c=0.53, CHCl₃)).

The term “C₁₋₈ carboxylic acid,” as used herein, refers tostraight-chained or branched, aromatic or non-aromatic, saturated orunsaturated, substituted or unsubstituted C₁₋₈ carboxylic acid,including, but not limited to, formic acid, acetic acid, propionic acid,n-butyric acid, t-butyric acid, n-pentanoic acid, 2-pentanoic acid,n-hexanoic acid, 2-hexanoic acid, n-heptanoic acid, n-octanoic acid,acrylic acid, succinic acid, fumaric acid, malic acid, tartaric acid,citric acid, lactic acid, and benzoic acid.

The term “C₁₋₈ sulfonic acid,” as used herein, refers tostraight-chained or branched, aromatic or non-aromatic, saturated orunsaturated, substituted or unsubstituted C₁₋₈ sulfonic acid, including,but not limited to, methanesulfonic acid, ethanesulfonic acid,n-propanesulfonic acid, 2-propanesulfonic acid, n-butanesulfonic acid,n-pentanesulfonic acid n-hexanesulfonic acid, n-heptanesulfonic acid,n-octanesulfonic acid, and benzenesulfonic acid.

The term “(−)-gossypol co-crystal,” as used herein, refers to acomposition comprising co-crystals of (−)-gossypol and a C₁₋₈ carboxylicacid or C₁₋₈ sulfonic acid.

The term “Bcl-2 family proteins,” as used herein, refers to both theanti-apoptotic members of the Bcl-2 family, including, but not limitedto, Bcl-2, Bcl-XL, Mcl-1, Al/BFL-1, BOO-DIVA, Bcl-w, Bcl-6, Bcl-8, andBcl-y, and the pro-apoptotic members of the Bcl-2 family, including, butnot limited to, Bak, Bax, Bad, tBid, Hrk, Bim, Bmf, as well as otherBcl-2 homology domain 3 (BH3) containing proteins that are regulated bygossypol compounds.

The term “overexpression of anti-apoptotic Bcl-2 family proteins,” asused herein, refers to an elevated level (e.g., aberrant level) of mRNAsencoding for an anti-apoptotic Bcl-2 family protein(s), and/or toelevated levels of anti-apoptotic Bcl-2 family protein(s) in cells ascompared to similar corresponding non-pathological cells expressingbasal levels of mRNAs encoding anti-apoptotic Bcl-2 family proteins orhaving basal levels of anti-apoptotic Bcl-2 family proteins. Methods fordetecting the levels of mRNAs encoding anti-apoptotic Bcl-2 familyproteins or levels of anti-apoptotic Bcl-2 family proteins in a cellinclude, but are not limited to, Western blotting using anti-apoptoticBcl-2 family protein antibodies, immunohistochemical methods, andmethods of nucleic acid amplification or direct RNA detection. Asimportant as the absolute level of anti-apoptotic Bcl-2 family proteinsin cells is to determining that they overexpress anti-apoptotic Bcl-2family proteins, so also is the relative level of anti-apoptotic Bcl-2family proteins to other pro-apoptotic signaling molecules (e.g.,pro-apoptotic Bcl-2 family proteins) within such cells. When the balanceof these two are such that, were it not for the levels of theanti-apoptotic Bcl-2 family proteins, the pro-apoptotic signalingmolecules would be sufficient to cause the cells to execute theapoptosis program and die, said cells would be dependent on theanti-apoptotic Bcl-2 family proteins for their survival. In such cells,exposure to an inhibiting effective amount of an anti-apoptotic Bcl-2family protein inhibitor will be sufficient to cause the cells toexecute the apoptosis program and die. Thus, the term “overexpression ofan anti-apoptotic Bcl-2 family protein” also refers to cells that, dueto the relative levels of pro-apoptotic signals and anti-apoptoticsignals, undergo apoptosis in response to inhibiting effective amountsof compounds that inhibit the function of anti-apoptotic Bcl-2 familyproteins.

The terms “anticancer agent” and “anticancer drug,” as used herein,refer to any therapeutic agent (e.g., chemotherapeutic compounds and/ormolecular therapeutic compounds), radiation therapies, or surgicalinterventions, used in the treatment of hyperproliferative diseases suchas cancer (e.g., in mammals).

The term “therapeutically effective amount,” as used herein, refers tothat amount of the therapeutic agent sufficient to result inamelioration of one or more symptoms of a disorder, or preventadvancement of a disorder, or cause regression of the disorder. Forexample, with respect to the treatment of cancer, a therapeuticallyeffective amount preferably refers to the amount of a therapeutic agentthat decreases the rate of tumor growth, decreases tumor mass, decreasesthe number of metastases, increases time to tumor progression, orincreases survival time by at least 5%, preferably at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 100%.

The terms “sensitize” and “sensitizing,” as used herein, refer tomaking, through the administration of a first agent (e.g., a compound ofFormula I), an animal or a cell within an animal more susceptible, ormore responsive, to the biological effects (e.g., promotion orretardation of an aspect of cellular function including, but not limitedto, cell growth, proliferation, invasion, angiogenesis, or apoptosis) ofa second agent. The sensitizing effect of a first agent on a target cellcan be measured as the difference in the intended biological effect(e.g., promotion or retardation of an aspect of cellular functionincluding, but not limited to, cell growth, proliferation, invasion,angiogenesis, or apoptosis) observed upon the administration of a secondagent with and without administration of the first agent. The responseof the sensitized cell can be increased by at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 100%, at least 150%, at least 200%, atleast 350%, at least 300%, at least 350%, at least 400%, at least 450%,or at least 500% over the response in the absence of the first agent.

The term “dysregulation of apoptosis,” as used herein, refers to anyaberration in the ability of (e.g., predisposition) a cell to undergocell death via apoptosis. Dysregulation of apoptosis is associated withor induced by a variety of conditions, including for example, autoimmunedisorders (e.g., systemic lupus erythematosus, rheumatoid arthritis,graft-versus-host disease, myasthenia gravis, or Sjögren's syndrome),chronic inflammatory conditions (e.g., psoriasis, asthma or Crohn'sdisease), hyperproliferative disorders (e.g., tumors, B cell lymphomas,or T cell lymphomas), viral infections (e.g., herpes, papilloma, orHIV), and other conditions such as osteoarthritis and atherosclerosis.It should be noted that when the dysregulation is induced by orassociated with a viral infection, the viral infection may or may not bedetectable at the time dysregulation occurs or is observed. That is,viral-induced dysregulation can occur even after the disappearance ofsymptoms of viral infection.

The term “hyperproliferative disease,” as used herein, refers to anycondition in which a localized population of proliferating cells in ananimal is not governed by the usual limitations of normal growth.Examples of hyperproliferative disorders include tumors, neoplasms,lymphomas and the like. A neoplasm is said to be benign if it does notundergo invasion or metastasis and malignant if it does either of these.A “metastatic” cell means that the cell can invade and destroyneighboring body structures. Hyperplasia is a form of cell proliferationinvolving an increase in cell number in a tissue or organ withoutsignificant alteration in structure or function. Metaplasia is a form ofcontrolled cell growth in which one type of fully differentiated cellsubstitutes for another type of differentiated cell.

The pathological growth of activated lymphoid cells often results in anautoimmune disorder or a chronic inflammatory condition. As used herein,the term “autoimmune disorder” refers to any condition in which anorganism produces antibodies or immune cells which recognize theorganism's own molecules, cells or tissues. Non-limiting examples ofautoimmune disorders include autoimmune hemolytic anemia, autoimmunehepatitis, Berger's disease or IgA nephropathy, celiac sprue, chronicfatigue syndrome, Crohn's disease, dermatomyositis, fibromyalgia, graftversus host disease, Grave's disease, Hashimoto's thyroiditis,idiopathic thrombocytopenia purpura, lichen planus, multiple sclerosis,myasthenia gravis, psoriasis, rheumatic fever, rheumatic arthritis,scleroderma, Sjögren's syndrome, systemic lupus erythematosus, type 1diabetes, ulcerative colitis, vitiligo, and the like.

The term “neoplastic disease,” as used herein, refers to any abnormalgrowth of cells being either benign (non-cancerous) or malignant(cancerous).

The term “anti-neoplastic agent,” as used herein, refers to any compoundthat retards the proliferation, growth, or spread of a targeted (e.g.,malignant) neoplasm.

The terms “prevent,” “preventing,” and “prevention,” as used herein,refer to a decrease in the occurrence of pathological cells (e.g.,hyperproliferative or neoplastic cells) in an animal. The prevention maybe complete, e.g., the total absence of pathological cells in a subject.The prevention may also be partial, such that the occurrence ofpathological cells in a subject is less than that which would haveoccurred without the present invention.

The term “synergistic,” as used herein, refers to an effect obtainedwhen (−)-gossypol co-crystal and a second agent are administeredtogether (e.g., at the same time or one after the other) that is greaterthan the additive effect of (−)-gossypol co-crystal and the second agentwhen administered individually. The synergistic effect allows for lowerdoses of (−)-gossypol co-crystal and/or the second agent to beadministered or provides greater efficacy at the same doses. Thesynergistic effect obtained can be at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 100%, at least 125%, at least 150%, at least175%, at least 200%, at least 250%, at least 300%, at least 350%, atleast 400%, or at least 500% more than the additive effect of the(−)-gossypol co-crystal compound and the second agent when administeredindividually. For example, with respect to the treatment of cancer, thesynergistic effect can be a decrease in the rate of tumor growth, adecrease in tumor mass, a decrease in the number of metastases, anincrease in time to tumor progression, or an increase in survival time.The co-administration of (−)-gossypol co-crystal and an anticancer agentmay allow for the use of lower doses of (−)-gossypol co-crystal and/orthe anticancer agent such that the cancer is effectively treated whileavoiding any substantial toxicity to the subject.

The term “about,” as used herein, includes the recited number +/−10%.Thus, “about 0.5” means 0.45 to 0.55.

The inhibitors of anti-apoptotic Bcl-2 family proteins of the presentinvention are compositions comprising co-crystals of (−)-gossypol with aC₁₋₈ carboxylic acid or C₁₋₈ sulfonic acid (“(−)-gossypol co-crystals”).(−)-Gossypol co-crystal is expected to be more stable than (−)-gossypolalone. Those skilled in the art will appreciate the importance ofcompound stability in the manufacturing, storage, shipping, and/orhandling of pharmaceutical compositions. The present compositions areexpected to be more stable than previously described compositionscomprising (−)-gossypol. Any C₁₋₈ carboxylic acid or C₁₋₈ sulfonic acidthat is capable of stabilizing (−)-gossypol can be used in theinvention. The molar ratio of (−)-gossypol to carboxylic acid orsulfonic acid in (−)-gossypol co-crystal ranges from about 10:1 to about1:10, preferably about 2:1 to about 1:2, more preferably about 1:1. Insome embodiments, the molar ratio of (−)-gossypol to carboxylic acid orsulfonic acid in (−)-gossypol co-crystal can be about 10:1, 9:1, 8:1,7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, or 1:10.

In one embodiment of the invention the C₁₋₈ carboxylic acid is aceticacid. In another embodiment, (−)-gossypol co-crystal comprises(−)-gossypol and acetic acid in a molar ratio of about 1:1. In apreferred embodiment, the 1:1 co-crystal of (−)-gossypol and acetic acidis in the form of yellow or pale yellow needle-shaped crystals. Inanother preferred embodiment, the co-crystal is characterized byintegration of ¹H NMR spectrum at δ 2.11 (s, 3H) which is one methylsignal of acetic acid and δ 2.18 (s, 6H) which is two methyl signals ofgossypol.

The compositions of this invention may be prepared using methods knownto those of skill in the art and as disclosed in the Examples. In oneembodiment, co-crystals are prepared by dissolving (−)-gossypol inacetone to form a solution, filtering the solution, adding a C₁₋₈carboxylic acid or C₁₋₈ sulfonic acid into the solution with mixinguntil the solution turns turbid, leaving the turbid solution at roomtemperature and then at reduced temperature to form co-crystals,collecting the co-crystals, washing the co-crystals with a solvent, anddrying the co-crystals. In one embodiment, the solution is mixed byconstant stirring. Reduced temperature is less than about 20° C.,preferably about 0-15° C., more preferably about 4° C. The time forco-crystal formation may range from 1 hour to 1 day; preferably the timeis about 1-4 hours. The co-crystals may be collected by any suitablemeans, including by filtration. The solvent for washing the co-crystalsmay be any suitable solvent, e.g., hexane, pentane, benzene, toluene, orpetroleum ether. The washed co-crystals may be dried at roomtemperature, preferably in a lightproof container. The co-crystals mayalso be dried in a vacuum drier, preferably at an elevated temperature(e.g., about 30-60° C., more preferably about 40° C.) for about 6-72hours, preferably about 12-48 hours.

(−)-Gossypol has been shown to bind to Bcl-2 and Bcl-X_(L) at the BH3binding groove and to have significant anticancer activity (U.S. PatentApplication No. 2003/0008924). An important aspect of the presentinvention is that (−)-gossypol co-crystal binds to and inhibitsanti-apoptotic Bcl-2 proteins in the same manner as gossypol. However,(−)-gossypol co-crystal is expected to be more stable than (−)-gossypol.Moreover, (−)-gossypol is a more potent inhibitor than racemic gossypol.Thus, compositions comprising (−)-gossypol co-crystal may be used toinduce apoptosis and also potentiate the induction of apoptosis inresponse to apoptosis induction signals. It is contemplated that thesecompositions sensitize cells to inducers of apoptosis, including cellsthat are resistant to such inducers. The compositions of the presentinvention can be used to induce apoptosis in any disorder that can betreated, ameliorated, or prevented by the induction of apoptosis. Thus,the present invention provides compositions and methods for targetinganimals characterized as overexpressing an anti-apoptotic Bcl-2 familyprotein. In some of the embodiments, the cells (e.g., cancer cells) showelevated expression levels of one or more anti-apoptotic Bcl-2 familyproteins as compared to non-pathological samples (e.g., non-cancerouscells). In other embodiments, the cells operationally manifest elevatedexpression levels of anti-apoptotic Bcl-2 family proteins by virtue ofexecuting the apoptosis program and dying in response to administrationof an inhibiting effective amount of (−)-gossypol co-crystal, saidresponse occurring, at least in part, due to the dependence in suchcells on anti-apoptotic Bcl-2 family protein function for theirsurvival.

In some embodiments, the compositions and methods of the presentinvention are used to treat diseased cells, tissues, organs, orpathological conditions and/or disease states in an animal (e.g., amammalian subject including, but not limited to, humans and veterinaryanimals). In this regard, various diseases and pathologies are amenableto treatment or prophylaxis using the present methods and compositions.A non-limiting exemplary list of these diseases and conditions includes,but is not limited to, cancers such as breast cancer, prostate cancer,lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma,malignant melanoma, ovarian cancer, brain cancer, primary braincarcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladdercancer, non-small cell lung cancer, head or neck carcinoma, breastcarcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma,Wilms' tumor, cervical carcinoma, testicular carcinoma, bladdercarcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma,prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma,esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma,renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma,malignant pancreatic insulinoma, malignant carcinoid carcinoma,choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervicalhyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocyticleukemia, acute myelogenous leukemia, chronic myelogenous leukemia,chronic granulocytic leukemia, acute granulocytic leukemia, hairy cellleukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma,polycythemia vera, essential thrombocytosis, Hodgkin's disease,non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primarymacroglobulinemia, and retinoblastoma, and the like; T and B cellmediated autoimmune diseases, inflammatory diseases, infections,hyperproliferative diseases, AIDS, degenerative conditions, vasculardiseases, and the like. In some embodiments, the cancer cells beingtreated are metastatic. In other embodiments, the cancer cells beingtreated are resistant to anticancer agents.

In some embodiments, infections suitable for treatment with thecompositions and methods of the present invention include, but are notlimited to, infections caused by viruses, bacteria, fungi, parasites,mycoplasma, prions, and the like.

Some embodiments of the present invention provide methods foradministering an effective amount of (−)-gossypol co-crystal and atleast one additional therapeutic agent (including, but not limited to,chemotherapeutic agents, antineoplastic agents, antimicrobial agents,antiviral agents, antifungal agents, and anti-inflammatory agents)and/or therapeutic technique (e.g., surgical intervention, and/orradiotherapies). In some embodiments, the combination of (−)-gossypolco-crystal and one or more therapeutic agents will have a greater effectas compared to the administration of either compound alone. In otherembodiments, the combination of (−)-gossypol co-crystal and one or moretherapeutic agents is expected to result in a synergistic effect (i.e.,more than additive) as compared to the administration of either onealone.

A number of suitable anticancer agents are contemplated for use in themethods of the present invention. Indeed, the present inventioncontemplates, but is not limited to, administration of numerousanticancer agents such as: agents that induce apoptosis; polynucleotides(e.g., anti-sense, ribozymes, siRNA); polypeptides (e.g., enzymes andantibodies); biological mimetics (e.g., gossypol or BH3 mimetics);agents that bind (e.g., oligomerize or complex) with a Bcl-2 familyprotein such as Bax; alkaloids; alkylating agents; antitumorantibiotics; antimetabolites; hormones; platinum compounds; monoclonalor polyclonal antibodies (e.g., antibodies conjugated with anticancerdrugs, toxins, defensins), toxins; radionuclides; biological responsemodifiers (e.g., interferons (e.g., IFN-α) and interleukins (e.g.,IL-2)); adoptive immunotherapy agents; hematopoietic growth factors;agents that induce tumor cell differentiation (e.g., all-trans-retinoicacid); gene therapy reagents (e.g., antisense therapy reagents andnucleotides); tumor vaccines; angiogenesis inhibitors; proteosomeinhibitors: NF-KB modulators; anti-CDK compounds; HDAC inhibitors; andthe like. Numerous other examples of chemotherapeutic compounds andanticancer therapies suitable for co-administration with the disclosedcompounds are known to those skilled in the art.

In preferred embodiments, anticancer agents comprise agents that induceor stimulate apoptosis. Agents that induce apoptosis include, but arenot limited to, radiation (e.g., X-rays, gamma rays, UV); kinaseinhibitors (e.g., epidermal growth factor receptor (EGFR) kinaseinhibitor, vascular growth factor receptor (VGFR) kinase inhibitor,fibroblast growth factor receptor (FGFR) kinase inhibitor,platelet-derived growth factor receptor (PDGFR) kinase inhibitor, andBcr-Abl kinase inhibitors (such as GLEEVEC)); antisense molecules;antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN, BEXXAR, and AVASTIN);anti-estrogens (e.g., raloxifene and tamoxifen); anti-androgens (e.g.,flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazole,and corticosteroids); cyclooxygenase 2 (COX-2) inhibitors (e.g.,celecoxib, meloxicam, NS-398, and non-steroidal anti-inflammatorydrugs); anti-inflammatory drugs (e.g., butazolidin, DECADRON, DELTASONE,dexamethasone, dexamethasone intensol, DEXONE, HEXADROL,hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, oxyphenbutazone,PEDIAPRED, phenylbutazone, PLAQUENIL, prednisolone, prednisone, PRELONE,and TANDEARIL); and cancer chemotherapeutic drugs (e.g., irinotecan(CAMPTOSAR), CPT-11, fludarabine (FLUDARA), dacarbazine, dexamethasone,mitoxantrone, MYLOTARG, VP-16, cisplatin, carboplatin, oxaliplatin,5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab,TAXOTERE or TAXOL); cellular signaling molecules; ceramides andcytokines; staurosporine, and the like.

In still other embodiments, the compositions and methods of the presentinvention provide (−)-gossypol co-crystal and at least oneanti-hyperproliferative or antineoplastic agent; e.g., selected fromalkylating agents, antimetabolites, and natural products (e.g., herbsand other plant and/or animal derived compounds).

Alkylating agents suitable for use in the present compositions andmethods include, but are not limited to: 1) nitrogen mustards (e.g.,mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin);and chlorambucil); 2) ethylenimines and methylmelamines (e.g.,hexamethylmelamine and thiotepa); 3) alkyl sulfonates (e.g., busulfan);4) nitrosoureas (e.g., carmustine (BCNU); lomustine (CCNU); semustine(methyl-CCNU); and streptozocin (streptozotocin)); and 5) triazenes(e.g., dacarbazine (dimethyltriazenoimid-azolecarboxamide).

In some embodiments, antimetabolites suitable for use in the presentcompositions and methods include, but are not limited to: 1) folic acidanalogs (e.g., methotrexate (amethopterin)); 2) pyrimidine analogs(e.g., fluorouracil (5-fluorouracil), floxuridine (fluorode-oxyuridine),and cytarabine (cytosine arabinoside)); and 3) purine analogs (e.g.,mercaptopurine (6-mercaptopurine), thioguanine (6-thioguanine), andpentostatin (2′-deoxycoformycin)).

In still further embodiments, chemotherapeutic agents suitable for usein the compositions and methods of the present invention include, butare not limited to: 1) vinca alkaloids (e.g., vinblastine, vincristine);2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics(e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin;rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), andmitomycin (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5)biological response modifiers (e.g., interferon-alfa); 6) platinumcoordinating complexes (e.g., cisplatin and carboplatin); 7)anthracenediones (e.g., mitoxantrone); 8) substituted ureas (e.g.,hydroxyurea); 9) methylhydrazine derivatives (e.g., procarbazine(N-methylhydrazine)); 10) adrenocortical suppressants (e.g., mitotane(o,p′-DDD) and aminoglutethimide); 11) adrenocorticosteroids (e.g.,prednisone); 12) progestins (e.g., hydroxyprogesterone caproate,medroxyprogesterone acetate, and megestrol acetate); 13) estrogens(e.g., diethylstilbestrol and ethinyl estradiol); 14) antiestrogens(e.g., tamoxifen); 15) androgens (e.g., testosterone propionate andfluoxymesterone); 16) antiandrogens (e.g., flutamide): and 17)gonadotropin-releasing hormone analogs (e.g., leuprolide).

Any oncolytic agent that is routinely used in a cancer therapy contextfinds use in the compositions and methods of the present invention. Forexample, the U.S. Food and Drug Administration maintains a formulary ofoncolytic agents approved for use in the United States. Internationalcounterpart agencies to the U.S.F.D.A. maintain similar formularies.Table 1 provides a list of exemplary antineoplastic agents approved foruse in the U.S. Those skilled in the art will appreciate that the“product labels” required on all U.S. approved chemotherapeuticsdescribe approved indications, dosing information, toxicity data, andthe like, for the exemplary agents.

TABLE 1 Aldesleukin Proleukin Chiron Corp., (des-alanyl-1, serine-125human interleukin-2) Emeryville, CA Alemtuzumab Campath Millennium andILEX (IgG1κ anti CD52 antibody) Partners, LP, Cambridge, MA AlitretinoinPanretin Ligand (9-cis-retinoic acid) Pharmaceuticals, Inc., San DiegoCA Allopurinol Zyloprim GlaxoSmithKline,(1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one Research Trianglemonosodium salt) Park, NC Altretamine Hexalen US Bioscience, West(N,N,N′,N′,N″,N″,-hexamethyl-1,3,5-triazine-2,4, Conshohocken, PA6-triamine) Amifostine Ethyol US Bioscience (ethanethiol,2-[(3-aminopropyl)amino]-, dihydrogen phosphate (ester)) AnastrozoleArimidex AstraZeneca (1,3-Benzenediacetonitrile, a, a, a′,a′-tetramethyl- Pharmaceuticals, LP, 5-(1H-1,2,4-triazoi-1-ylmethyl))Wilmington, DE Arsenic trioxide Trisenox Cell Therapeutic, Inc.,Seattle, WA Asparaginase Elspar Merck & Co., Inc., (L-asparagineamidohydrolase, type EC-2) Whitehouse Station, NJ BCG Live TICE BCGOrganon Teknika, (lyophilized preparation of an attenuated strain ofCorp., Durham, NC Mycobacterium bovis (Bacillus Calmette-Gukin [BCG],substrain Montreal) bexarotene capsules Targretin Ligand(4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2- Pharmaceuticalsnapthalenyl) ethenyl] benzoic acid) bexarotene gel Targretin LigandPharmaceuticals Bleomycin Blenoxane Bristol-Myers Squibb (cytotoxicglycopeptide antibiotics produced by Co., NY, NY Streptomycesverticillus; bleomycin A₂ and bleomycin B₂) Capecitabine Xeloda Roche(5′-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]- cytidine) CarboplatinParaplatin Bristol-Myers Squibb (platinum, diammine [1,1-cyclobutanedicarboxylato(2-)-0,0′]-,(SP-4-2)) Carmustine BCNU, BiCNUBristol-Myers Squibb (1,3-bis(2-chloroethyl)-1-nitrosourea) Carmustinewith Polifeprosan 20 Implant Gliadel Wafer Guilford Pharmaceuticals,Inc., Baltimore, MD Celecoxib Celebrex Searle (as4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H- Pharmaceuticals,pyrazol-1-yl] England benzenesulfonamide) Chlorambucil LeukeranGlaxoSmithKline (4-[bis(2chlorethyl)amino]benzenebutanoic acid)Cisplatin Platinol Bristol-Myers Squibb (PtCl₂H₆N₂) CladribineLeustatin, 2-CdA R. W. Johnson (2-chloro-2′-deoxy-b-D-adenosine)Pharmaceutical Research Institute, Raritan, NJ Cyclophosphamide Cytoxan,Neosar Bristol-Myers Squibb (2-[bis(2-chloroethyl)amino]tetrahydro-2H-13,2- oxazaphosphorine 2-oxide monohydrate) CytarabineCytosar-U Pharmacia & Upjohn (1-b-D-Arabinofuranosylcytosine, C₉H₁₃N₃O₅)Company cytarabine liposomal DepoCyt Skye Pharmaceuticals, Inc., SanDiego, CA Dacarbazine DTIC-Dome Bayer AG,(5-(3,3-dimethyl-1-triazeno)-imidazole-4- Leverkusen, Germanycarboxamide (DTIC)) Dactinomycin, actinomycin D Cosmegen Merck(actinomycin produced by Streptomyces parvullus, C₆₂H₈₆N₁₂O₁₆)Darbepoetin alfa Aranesp Amgen, Inc., (recombinant peptide) ThousandOaks, CA daunorubicin liposomal DanuoXome Nexstar((8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-á- Pharmaceuticals, Inc.,L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro- Boulder, CO6,8,11-trihydroxy-1-methoxy-5,12- naphthacenedione hydrochloride)Daunorubicin HCl, daunomycin Cerubidine Wyeth Ayerst, ((1 S ,3 S)-3-Acetyl-1,2,3,4,6,11-hexahydro- Madison, NJ3,5,12-trihydroxy-10-methoxy-6,11-dioxo-1- naphthacenyl3-amino-2,3,6-trideoxy-(alpha)-L- lyxo -hexopyranoside hydrochloride)Denileukin diftitox Ontak Seragen, Inc., (recombinant peptide)Hopkinton, MA Dexrazoxane Zinecard Pharmacia & Upjohn((S)-4,4′-(1-methyl-1,2-ethanediyl)bis-2,6- Company piperazinedione)Docetaxel Taxotere Aventis ((2R,3S)-N-carboxy-3-phenylisoserine, N-tert-Pharmaceuticals, Inc., butyl ester, 13-ester with 5b-20-epoxy-Bridgewater, NJ 12a,4,7b,10b,13a-hexahydroxytax-11-en-9-one 4- acetate2-benzoate, trihydrate) Doxorubicin HCl Adriamycin, Pharmacia & Upjohn(8S,10S)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo- Rubex Companyhexopyranosyl)oxy]-8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12- naphthacenedionehydrochloride) doxorubicin Adriamycin PFS Pharmacia & Upjohn IntravenousCompany injection doxorubicin liposomal Doxil Sequus Pharmaceuticals,Inc., Menlo park, CA dromostanolone propionate Dromostanolone Eli Lilly& Company, (17b-Hydroxy-2a-methyl-5a-androstan-3-one Indianapolis, INpropionate) dromostanolone propionate Masterone Syntex, Corp., Paloinjection Alto, CA Elliott′s B Solution Elliott′s B Orphan Medical, IncSolution Epirubicin Ellence Pharmacia & Upjohn((8S-cis)-10-[(3-amino-2,3,6-trideoxy-a-L-arabino- Companyhexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12- naphthacenedionehydrochloride) Epoetin alfa Epogen Amgen, Inc (recombinant peptide)Estramustine Emcyt Pharmacia & Upjohn(estra-1,3,5(10)-triene-3,17-diol(17(beta))-,3- Company[bis(2-chloroethyl)carbamate] 17-(dihydrogen phosphate), disodium salt,monohydrate, or estradiol 3-[bis(2-chloroethyl)carbamate] 17-(dihydrogen phosphate), disodium salt, monohydrate) Etoposide phosphateEtopophos Bristol-Myers Squibb (4′-Demethylepipodophyllotoxin9-[4,6-O-(R)- ethylidene-(beta)-D-glucopyranoside],4′- (dihydrogenphosphate)) etoposide, VP-16 Vepesid Bristol-Myers Squibb(4′-demethylepipodophyllotoxin 9-[4,6-0-(R)-ethylidene-(beta)-D-glucopyranoside]) Exemestane Aromasin Pharmacia &Upjohn (6-methylenandrosta-1,4-diene-3,17-dione) Company FilgrastimNeupogen Amgen, Inc (r-metHuG-CSF) floxuridine (intraarterial) FUDRRoche (2′-deoxy-5-fluorouridine) Fludarabine Fludara BerlexLaboratories, (fluorinated nucleotide analog of the antiviral agentInc., Cedar Knolls, NJ vidarabine, 9-b-D-arabinofuranosyladenine (ara-A)) Fluorouracil, 5-FU Adrucil ICN Pharmaceuticals,(5-fluoro-2,4(1H,3H)-pyrimidinedione) Inc., Humacao, Puerto RicoFulvestrant Faslodex IPR Pharmaceuticals, (7-alpha-[9-(4,4,5,5,5-pentafluoropentylsulphinyl) Guayama, Puertononyl]estra-1,3,5-(10)-triene-3,17-beta-diol) Rico Gemcitabine GemzarEli Lilly (2′-deoxy-2′, 2′-difluorocytidine monohydrochloride(b-isomer)) Gemtuzumab Ozogamicin Mylotarg Wyeth Ayerst (anti-CD33hP67.6) Goserelin acetate Zoladex Implant AstraZeneca (acetate salt of[D-Ser(But)⁶,Azgly¹⁰]LHRH; pyro- PharmaceuticalsGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro- Azgly-NH2 acetate[C₅₉H₈₄N₁₈O₁₄.(C₂H₄O₂)_(x) Hydroxyurea Hydrea Bristol-Myers SquibbIbritumomab Tiuxetan Zevalin Biogen IDEC, Inc., (immunoconjugateresulting from a thiourea Cambridge MA covalent bond between themonoclonal antibody Ibritumomab and the linker-chelator tiuxetan [N-[2-bis(carboxymethyl)amino]-3-(p- isothiocyanatophenyl)-propyl]-[N-[2-bis(carboxymethyl)amino]-2-(methyl)- ethyl]glycine) Idarubicin IdamycinPharmacia & Upjohn (5,12-Naphthacenedione,9-acetyl-7-[(3-amino- Company2,3,6-trideoxy-(alpha)-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,9,11-trihydroxyhydrochloride,(7S-cis )) Ifosfamide IFEX Bristol-Myers Squibb(3-(2-chloroethyl)-2-[(2- chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide) Imatinib Mesilate Gleevec Novartis AG, Basel,(4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl- Switzerland3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]- phenyl]benzamidemethanesulfonate) Interferon alfa-2a Roferon-A Hoffrnann-La Roche,(recombinant peptide) Inc., Nutley, NJ Interferon alfa-2b Intron ASchering AG, Berlin, (recombinant peptide) (Lyophilized GermanyBetaseron) Irinotecan HCl Camptosar Pharmacia & Upjohn((4S)-4,11-diethyl-4-hydroxy-9-[(4-piperi- Companydinopiperidino)carbonyloxy]-1H-pyrano[3′,4′:6,7] indolizino[1,2-b]quinoline-3,14(4H,12H) dione hydrochloride trihydrate) Letrozole FemaraNovartis (4,4′-(1H-1,2,4-Triazol-1-ylmethylene) dibenzonitrile)Leucovorin Wellcovorin, Immunex, Corp., (L-Glutamic acid,N[4[[(2amino-5-formyl- Leucovorin Seattle, WA 1,4,5,6,7,8hexahydro4oxo6- pteridinyl)methyl]amino]benzoyl], calcium salt (1:1))Levamisole HCl Ergamisol Janssen Research((−)-(S)-2,3,5,6-tetrahydro-6-phenylimidazo [2,1- Foundation, b]thiazole monohydrochloride C₁₁H₁₂N₂S.HCl) Titusville, NJ Lomustine CeeNUBristol-Myers Squibb (1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea)Meclorethamine, nitrogen mustard Mustargen Merck(2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride) Megestrolacetate Megace Bristol-Myers Squibb17α(acetyloxy)-6-methylpregna-4,6-diene- 3,20-dione Melphalan, L-PAMAlkeran GlaxoSmithKline (4-[bis(2-chloroethyl) amino]-L-phenylalanine)Mercaptopurine, 6-MP Purinethol GlaxoSmithKline(1,7-dihydro-6H-purine-6-thione monohydrate) Mesna Mesnex Asta Medica(sodium 2-mercaptoethane sulfonate) Methotrexate Methotrexate LederleLaboratories (N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L- glutamic acid) MethoxsalenUvadex Therakos, Inc., Way(9-methoxy-7H-furo[3,2-g][1]-benzopyran-7-one) Exton, Pa Mitomycin CMutamycin Bristol-Myers Squibb mitomycin C Mitozytrex SuperGen, Inc.,Dublin, CA Mitotane Lysodren Bristol-Myers Squibb(1,1-dichloro-2-(o-chlorophenyl)-2-(p- chlorophenyl) ethane)Mitoxantrone Novantrone Immunex Corporation(1,4-dihydroxy-5,8-bis[[2-[(2- hydroxyethyl)amino]ethyl]amino]-9,10-anthracenedione dihydrochloride) Nandrolone phenpropionate Durabolin-50Organon, Inc., West Orange, NJ Nofetumomab Verluma Boehringer IngelheimPharma KG, Germany Oprelvekin Neumega Genetics Institute, (IL-11) Inc.,Alexandria, VA Oxaliplatin Eloxatin Sanofi Synthelabo,(cis-[(1R,2R)-1,2-cyclohexanediamine-N,N′] Inc., NY, NY[oxalato(2-)-O,O′] platinum) Paclitaxel TAXOL Bristol-Myers Squibb (5β,20-Epoxy-1,2a,4,7β,10β,13a- hexahydroxytax-11-en-9-one 4,10-diacetate 2-benzoate 13-ester with (2R,3S)-N-benzoyl-3- phenylisoserine) PamidronateAredia Novartis (phosphonic acid (3-amino-1-hydroxypropylidene) bis-,disodium salt, pentahydrate, (APD)) Pegademase Adagen Enzon((monomethoxypolyethylene glycol succinimidyl) (PegademasePharmaceuticals, Inc., 11-17-adenosine deaminase) Bovine) Bridgewater,NJ Pegaspargase Oncaspar Enzon (monomethoxypolyethylene glycolsuccinimidyl L- asparaginase) Pegfilgrastim Neulasta Amgen, Inc(covalent conjugate of recombinant methionyl human G-CSF (Filgrastim)and monomethoxypolyethylene glycol) Pentostatin Nipent Parke-DavisPharmaceutical Co., Rockville, MD Pipobroman Vercyte AbbottLaboratories, Abbott Park, IL Plicamycin, Mithramycin Mithracin Pfizer,Inc., NY, NY (antibiotic produced by Streptomyces plicatus) Porfimersodium Photofrin QLT Phototherapeutics, Inc., Vancouver, CanadaProcarbazine Matulane Sigma Tau(N-isopropyl-μ-(2-methylhydrazino)-p-toluamide Pharmaceuticals, Inc.,monohydrochloride) Gaithersburg, MD Quinacrine Atabrine Abbott Labs(6-chloro-9-(1-methyl-4-diethyl-amine) butylamino-2-methoxyacridine)Rasburicase Elitek Sanofi-Synthelabo, (recombinant peptide) Inc.,Rituximab Rituxan Genentech, Inc., (recombinant anti-CD20 antibody)South San Francisco, CA Sargramostim Prokine Immunex Corp (recombinantpeptide) Streptozocin Zanosar Pharmacia & Upjohn (streptozocin2-deoxy-2- Company [[(methylnitrosoamino)carbonyl]amino]-a(and b)-D-glucopyranose and 220 mg citric acid anhydrous) Talc Sclerosol Bryan,Corp., (Mg₃Si₄O₁₀(OH)₂) Woburn, MA Tamoxifen Nolvadex AstraZeneca((Z)2-[4-(1,2-diphenyl-1-butenyl) phenoxy]-N,N- Pharmaceuticalsdimethylethanamine 2-hydroxy-1,2,3- propanetricarboxylate (1:1))Temozolomide Temodar Schering(3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as- tetrazine-8-carboxamide)teniposide, VM-26 Vumon Bristol-Myers Squibb(4′-demethylepipodophyllotoxin 9-[4,6-0-(R)-2-thenylidene-(beta)-D-glucopyranoside]) Testolactone Teslac Bristol-MyersSquibb (13-hydroxy-3-oxo-13,17-secoandrosta-1,4-dien- 17-oic acid[dgr]-lactone) Thioguanine, 6-TG Thioguanine GlaxoSmithKline(2-amino-1,7-dihydro-6H-purine-6-thione) Thiotepa Thioplex ImmunexCorporation (Aziridine, 1,1′,1″-phosphinothioylidynetris-, or Tris(1-aziridinyl) phosphine sulfide) Topotecan HCl Hycamtin GlaxoSmithKline((S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7] indolizino [1,2-b]quinoline-3,14-(4H,12H)-dione monohydrochloride) Toremifene FarestonRoberts (2-(p-[(Z)-4-chloro-1,2-diphenyl-1-butenyl]- PharmaceuticalCorp., phenoxy)-N,N-dimethylethylamine citrate (1:1)) Eatontown, NJTositumomab, I 131 Tositumomab Bexxar Corixa Corp., Seattle,(recombinant murine immunotherapeutic WA monoclonal IgG_(2a) lambdaanti-CD20 antibody (I 131 is a radioimmunotherapeutic antibody))Trastuzumab Herceptin Genentech, Inc (recombinant monoclonal IgG₁ kappaanti-HER2 antibody) Tretinoin, ATRA Vesanoid Roche (all-trans retinoicacid) Uracil Mustard Uracil Mustard Roberts Labs Capsules Valrubicin,N-trifluoroacetyladriamycin-14- Valstar Anthra --> Medeva valerate((2S-cis)-2-[1,2,3,4,6,11-hexahydro-2,5,12- trihydroxy-7methoxy-6,11-dioxo-[[4 2,3,6-trideoxy-3-[(trifluoroacetyl)-amino-α-L-lyxo-hexopyranosyl]oxyl]-2-naphthacenyl]-2-oxoethyl pentanoate) Vinblastine,Leurocristine Velban Eli Lilly (C₄₆H₅₆N₄O₁₀.H₂SO₄) Vincristine OncovinEli Lilly (C₄₆H₅₆N₄O₁₀.H₂SO₄) Vinorelbine Navelbine GlaxoSmithKline(3′,4′-didehydro-4′-deoxy-C′- norvincaleukoblastine[R-(R*,R*)-2,3-dihydroxybutanedioate (1:2)(salt)]) Zoledronate, Zoledronic acid ZometaNovartis ((1-Hydroxy-2-imidazol-1-yl-phosphonoethyl) phosphonic acidmonohydrate)

Preferred conventional anticancer agents for use in administration withthe present compounds include, but are not limited to, adriamycin,5-fluorouracil, etoposide, camptothecin, actinomycin D, mitomycin C,cisplatin, docetaxel, gemcitabine, carboplatin, oxaliplatin, bortezomib,gefitinib, and bevacizumab. These agents can be prepared and usedsingularly, in combined therapeutic compositions, in kits, or incombination with immunotherapeutic agents, and the like.

For a more detailed description of anticancer agents and othertherapeutic agents, those skilled in the art are referred to any numberof instructive manuals including, but not limited to, the Physician'sDesk Reference and to Goodman and Gilman's “Pharmaceutical Basis ofTherapeutics” ninth edition, Eds. Hardman et al., 1996.

The present invention provides methods for administering (−)-gossypolco-crystal with radiation therapy. The invention is not limited by thetypes, amounts, or delivery and administration systems used to deliverthe therapeutic dose of radiation to an animal. For example, the animalmay receive photon radiotherapy, particle beam radiation therapy,radioisotope therapy (e.g., radioconjugates with monoclonal antibodies),other types of radiotherapies, and combinations thereof. In someembodiments, the radiation is delivered to the animal using a linearaccelerator. In still other embodiments, the radiation is deliveredusing a gamma knife.

The source of radiation can be external or internal to the animal.External radiation therapy is most common and involves directing a beamof high-energy radiation to a tumor site through the skin using, forinstance, a linear accelerator. While the beam of radiation is localizedto the tumor site, it is nearly impossible to avoid exposure of normal,healthy tissue. However, external radiation is usually well tolerated bypatients. Internal radiation therapy involves implanting aradiation-emitting source, such as beads, wires, pellets, capsules,particles, and the like, inside the body at or near the tumor siteincluding the use of delivery systems that specifically target cancercells (e.g., using particles attached to cancer cell binding ligands).Such implants can be removed following treatment, or left in the bodyinactive. Types of internal radiation therapy include, but are notlimited to, brachytherapy, interstitial irradiation, intracavityirradiation, radioimmunotherapy, and the like.

The animal may optionally receive radiosensitizers (e.g., metronidazole,misonidazole, intra-arterial Budr, intravenous iododeoxyuridine (IudR),nitroimidazole, 5-substituted-4-nitroimidazoles, 2H-isoindolediones,[[(2-bromoethyl)-amino]methyl]-nitro-1H-imidazole-1-ethanol,nitroaniline derivatives, DNA-affinic hypoxia selective cytotoxins,halogenated DNA ligand, 1,2,4 benzotriazine oxides, 2-nitroimidazolederivatives, fluorine-containing nitroazole derivatives, benzamide,nicotinamide, acridine-intercalator, 5-thiotretrazole derivative,3-nitro-1,2,4-triazole, 4,5-dinitroimidazole derivative, hydroxylatedtexaphrins, cisplatin, mitomycin, tiripazamine, nitrosourea,mercaptopurine, methotrexate, fluorouracil, bleomycin, vincristine,carboplatin, epirubicin, doxorubicin, cyclophosphamide, vindesine,etoposide, paclitaxel, heat (hyperthermia), and the like),radioprotectors (e.g., cysteamine, aminoalkyl dihydrogenphosphorothioates, amifostine (WR 2721), IL-1, IL-6, and the like).Radiosensitizers enhance the killing of tumor cells. Radioprotectorsprotect healthy tissue from the harmful effects of radiation.

Any type of radiation can be administered to a patient, so long as thedose of radiation is tolerated by the patient without unacceptablenegative side-effects. Suitable types of radiotherapy include, forexample, ionizing (electromagnetic) radiotherapy (e.g., X-rays or gammarays) or particle beam radiation therapy (e.g., high linear energyradiation). Ionizing radiation is defined as radiation comprisingparticles or photons that have sufficient energy to produce ionization,i.e., gain or loss of electrons (as described in, for example, U.S. Pat.No. 5,770,581 incorporated herein by reference in its entirety). Theeffects of radiation can be at least partially controlled by theclinician. The dose of radiation is preferably fractionated for maximaltarget cell exposure and reduced toxicity.

The total dose of radiation administered to an animal preferably isabout 0.01 Gray (Gy) to about 100 Gy. More preferably, about 10 Gy toabout 65 Gy (e.g., about 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45Gy, 50 Gy, 55 Gy, or 60 Gy) are administered over the course oftreatment. While in some embodiments a complete dose of radiation can beadministered over the course of one day, the total dose is ideallyfractionated and administered over several days. Desirably, radiotherapyis administered over the course of at least about 3 days, e.g., at least5, 7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52, or 56 days (about1-8 weeks). Accordingly, a daily dose of radiation will compriseapproximately 1-5 Gy (e.g., about 1 Gy, 1.5 Gy, 1.8 Gy, 2 Gy, 2.5 Gy,2.8 Gy, 3 Gy, 3.2 Gy, 3.5 Gy, 3.8 Gy, 4 Gy, 4.2 Gy, or 4.5 Gy),preferably 1-2 Gy (e.g., 1.5-2 Gy). The daily dose of radiation shouldbe sufficient to induce destruction of the targeted cells. If stretchedover a period, radiation preferably is not administered every day,thereby allowing the animal to rest and the effects of the therapy to berealized. For example, radiation desirably is administered on 5consecutive days, and not administered on 2 days, for each week oftreatment, thereby allowing 2 days of rest per week. However, radiationcan be administered 1 day/week, 2 days/week, 3 days/week, 4 days/week, 5days/week, 6 days/week, or all 7 days/week, depending on the animal'sresponsiveness and any potential side effects. Radiation therapy can beinitiated at any time in the therapeutic period. Preferably, radiationis initiated in week 1 or week 2, and is administered for the remainingduration of the therapeutic period. For example, radiation isadministered in weeks 1-6 or in weeks 2-6 of a therapeutic periodcomprising 6 weeks for treating, for instance, a solid tumor.Alternatively, radiation is administered in weeks 1-5 or weeks 2-5 of atherapeutic period comprising 5 weeks. These exemplary radiotherapyadministration schedules are not intended, however, to limit the presentinvention.

Antimicrobial therapeutic agents may also be used as therapeutic agentsin the present invention. Any agent that can kill, inhibit, or otherwiseattenuate the function of microbial organisms may be used, as well asany agent contemplated to have such activities. Antimicrobial agentsinclude, but are not limited to, natural and synthetic antibiotics,antibodies, inhibitory proteins (e.g., defensins), antisense nucleicacids, membrane disruptive agents and the like, used alone or incombination. Indeed, any type of antibiotic may be used including, butnot limited to, antibacterial agents, antiviral agents, antifungalagents, and the like.

In some embodiments of the present invention, (−)-gossypol co-crystaland one or more therapeutic agents or anticancer agents are administeredto an animal under one or more of the following conditions: at differentperiodicities, at different durations, at different concentrations, bydifferent administration routes, etc. In some embodiments, (−)-gossypolco-crystal is administered prior to the therapeutic or anticancer agent,e.g., 0.5, 1, 2 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days,1, 2, 3, or 4 weeks prior to the administration of the therapeutic oranticancer agent. In some embodiments, (−)-gossypol co-crystal isadministered after the therapeutic or anticancer agent, e.g., 0.5, 1, 23, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, 1, 2, 3, or 4weeks after the administration of the anticancer agent. In someembodiments, (−)-gossypol co-crystal and the therapeutic or anticanceragent are administered concurrently but on different schedules, e.g.,(−)-gossypol co-crystal is administered daily while the therapeutic oranticancer agent is administered once a week, once every two weeks, onceevery three weeks, or once every four weeks. In other embodiments,(−)-gossypol co-crystal is administered once a week while thetherapeutic or anticancer agent is administered daily, once a week, onceevery two weeks, once every three weeks, or once every four weeks.

Pharmaceutical compositions can be produced by combining (−)-gossypolco-crystal in a therapeutically effective amount to induce apoptosis incells or to sensitize cells to inducers of apoptosis with apharmaceutically acceptable carrier. The novel pharmaceuticalcompositions of the present invention comprise intact (−)-gossypolco-crystal. In some embodiments, the pharmaceutical compositionscomprise (−)-gossypol co-crystal in combination with a liquid in whichthe co-crystal is substantially insoluble (e.g., water) such that asuspension is formed.

Compositions within the scope of this invention include all compositionswherein the compositions of the present invention are contained in anamount which is effective to achieve its intended purpose. Whileindividual needs vary, determination of optimal ranges of effectiveamounts of each component is within the skill of the art. Typically, thecompositions may be administered to mammals, e.g. humans, orally at adose of 0.0025 to 50 mg/kg, or an equivalent amount of thepharmaceutically acceptable salt thereof, per day of the body weight ofthe mammal being treated for disorders responsive to induction ofapoptosis. Preferably, about 0.01 to about 10 mg/kg is orallyadministered to treat, ameliorate, or prevent such disorders. Forintramuscular injection, the dose is generally about one-half of theoral dose. For example, a suitable intramuscular dose would be about0.0025 to about 25 mg/kg, and most preferably, from about 0.01 to about5 mg/kg.

The unit oral dose may comprise from about 0.01 to about 200 mg,preferably about 0.1 to about 100 mg of the composition. The unit dosemay be administered one or more times daily as one or more tablets orcapsules each containing from about 0.1 to about 100 mg, convenientlyabout 0.25 to 50 mg of the composition.

In a topical formulation, the composition may be present at aconcentration of about 0.01 to 100 mg per gram of carrier. In apreferred embodiment, the composition is present at a concentration ofabout 0.07-1.0 mg/ml, more preferably, about 0.1-0.5 mg/ml, mostpreferably, about 0.4 mg/ml.

In addition to administering (−)-gossypol co-crystal as a raw chemical,the compositions of the invention may be administered as part of apharmaceutical preparation containing suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the compositions into preparations which can beused pharmaceutically. Preferably, the preparations, particularly thosepreparations which can be administered orally or topically and which canbe used for the preferred type of administration, such as tablets,dragees, slow release lozenges and capsules, mouth rinses and mouthwashes, gels, liquid suspensions, hair rinses, hair gels, shampoos andalso preparations which can be administered rectally, such assuppositories, as well as suitable solutions for administration byinjection, topically or orally, contain from about 0.01 to 99 percent,preferably from about 0.25 to 75 percent of active compound(s), togetherwith the excipient.

The pharmaceutical compositions of the invention may be administered toany animal which may experience the beneficial effects of the compoundsof the invention. Foremost among such animals are mammals, e.g., humans,although the invention is not intended to be so limited. Other animalsinclude veterinary animals (cows, sheep, pigs, horses, dogs, cats andthe like).

The compositions and pharmaceutical compositions thereof may beadministered by any means that achieve their intended purpose. Forexample, administration may be by parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, buccal, intrathecal,intracranial, intranasal, or topical routes. Alternatively, orconcurrently, administration may be by the oral route. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides, forexample lactose or sucrose, mannitol or sorbitol, cellulose preparationsand/or calcium phosphates, for example tricalcium phosphate or calciumhydrogen phosphate, as well as binders such as starch paste, using, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose,sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariesare, above all, flow-regulating agents and lubricants, for example,silica, talc, stearic acid or salts thereof, such as magnesium stearateor calcium stearate, and/or polyethylene glycol. Dragee cores areprovided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated saccharide solutions maybe used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquersolutions and suitable organic solvents or solvent mixtures. In order toproduce coatings resistant to gastric juices, solutions of suitablecellulose preparations such as acetylcellulose phthalate orhydroxypropylmethyl-cellulose phthalate, are used. Dye stuffs orpigments may be added to the tablets or dragee coatings, for example,for identification or in order to characterize combinations of activecompound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules whichmay be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, orliquid paraffin. In addition, stabilizers may be added.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories, which consist of a combination of one ormore of the active compounds with a suppository base. Suitablesuppository bases are, for example, natural or synthetic triglycerides,or paraffin hydrocarbons. In addition, it is also possible to usegelatin rectal capsules which consist of a combination of the activecompounds with a base. Possible base materials include, for example,liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts and alkaline solutions. In addition, suspensions ofthe active compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, ethyl oleate or triglycerides or polyethylene glycol-400.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension include, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may alsocontain stabilizers.

The topical compositions of this invention are formulated preferably asoils, creams, lotions, ointments and the like by choice of appropriatecarriers. Suitable carriers include vegetable or mineral oils, whitepetrolatum (white soft paraffin), branched chain fats or oils, animalfats and high molecular weight alcohol (greater than C₁₂). The preferredcarriers are those in which the active ingredient is soluble.Emulsifiers, stabilizers, humectants and antioxidants may also beincluded as well as agents imparting color or fragrance, if desired.Additionally, transdermal penetration enhancers can be employed in thesetopical formulations. Examples of such enhancers can be found in U.S.Pat. Nos. 3,989,816 and 4,444,762.

Creams are preferably formulated from a mixture of mineral oil,self-emulsifying beeswax and water in which mixture the activeingredient, dissolved in a small amount of an oil such as almond oil, isadmixed. A typical example of such a cream is one which includes about40 parts water, about 20 parts beeswax, about 40 parts mineral oil andabout 1 part almond oil.

Ointments may be formulated by mixing a suspension of the activeingredient in a vegetable oil such as almond oil with warm soft paraffinand allowing the mixture to cool. A typical example of such an ointmentis one which includes about 30% almond oil and about 70% white softparaffin by weight.

Lotions may be conveniently prepared by preparing a suspension of theactive ingredient in a suitable high molecular weight alcohol such aspropylene glycol or polyethylene glycol.

The following examples are illustrative, but not limiting, of the methodand compositions of the present invention. Other suitable modificationsand adaptations of the variety of conditions and parameters normallyencountered in clinical therapy and which are obvious to those skilledin the art are within the spirit and scope of the invention.

EXAMPLE 1 Preparation of (−)-Gossypol Acetic Acid Co-Crystal

All chemicals and reagents were purchased from Aldrich Chemical Co. orLancaster Synthesis Inc. and used without further purification.(−)-Gossypol (1 g) was dissolved in acetone (6 ml) and filtered. Aceticacid was added into the constantly stirred filtrate until the solutionturned turbid. The mixture was left at room temperature for 2 hours andthen at 4° C. for 2 hours. The co-crystals were collected by filtrationusing a Buchner funnel under reduced pressure and washed with a smallamount of hexane. Pure (−)-gossypol acetic acid was first dried in alightproof container and further dried in a vacuum drier at 40° C. for24 hours.

EXAMPLE 2 Characterization of (−)-Gossypol Acetic Acid Co-Crystals

(−)-Gossypol acetic acid co-crystals were yellow or pale yellow andneedle shaped. The co-crystals were readily soluble in acetone andether, slightly soluble in chloroform and ethanol, and sparsely solublein petroleum. The co-crystals were insoluble in water. The uncorrectedmelting point of the co-crystals was determined to be 178-180° C. usinga Mel-Temp apparatus.

¹H and ¹³C nuclear magnetic resonance (NMR) spectra of the co-crystals(FIGS. 1 and 2) were recorded on a Bruker 300 instrument. Samples weredissolved in an appropriate deuterated solvent (CDCl₃). Proton chemicalshifts were reported as parts per million (δ) relative totetramethylsilane (0.00 ppm), which was used as an internal standard.Chemical shifts for ¹³C NMR spectra were reported as δ relative todeuterated chloroform (CDCl₃, 77.0 ppm). ¹H NMR (300 MHz, CDCl₃) δ 15.21(s, 2H), 11.16 (s, 2H), 7.80 (s, 2H), 6.45 (s, 2H), 5.79 (s, 2H),4.08-3.80 (m, 2H), 2.18 (s, 6H), 2.11 (s, 3H), 1.58 (d, J=6.8 Hz, 12H).¹³C NMR (75 MHz, CDCl₃) δ 199.4, 176.8, 156.0, 150.5, 143.4, 134.1,133.7, 129.7, 118.1, 115.9, 114.6, 111.8, 27.9, 20.7, 20.3, 20.2. Basedon the ¹H NMR spectrum, the co-crystal was determined to be a complex of(−)-gossypol with acetic acid at a molar ratio of 1:1.

The infrared spectrum (FIG. 3) of the co-crystals was recorded on aPerkin-Elmer FT-IR spectrometer. IR(KBr) 3421, 2959, 2929, 1710, 1611,1577, 1440, 1379, 1339, 1269, 1176, 1052, 841, 772 cm⁻¹.

The electrospray mass spectrum (FIG. 4) of the co-crystals was performedon a Micromass AutoSpec Ultima Magnetic sector mass spectrometer. MS m/z541 (M+Na)⁺.

The X-ray powder diffraction spectrum (FIG. 5) of the co-crystals wasrecorded on a Scintag X-ray powder diffractometer. Based on thespectrum, the co-crystal was determined to be a complex of (−)-gossypolwith acetic acid at a molar ratio of 1:1.

Having now fully described the invention, it will be understood by thoseof skill in the art that the same can be performed within a wide andequivalent range of conditions, formulations, and other parameterswithout affecting the scope of the invention or any embodiment thereof.All patents, patent applications and publications cited herein are fullyincorporated by reference herein in their entirety.

1. A composition consisting essentially of co-crystals of (−)-gossypol with acetic acid in a molar ratio of about 1:1.
 2. The composition of claim 1, which is characterized by integration of ¹H NMR spectrum at δ2.11 (s, 3H) which is one methyl signal of acetic acid and δ 2.18 (s, 6H) which is two methyl signals of gossypol.
 3. A kit comprising the co-crystals of claim 1 and instructions for administering said composition to an animal.
 4. The kit of claim 3, wherein said composition is in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
 5. The kit of claim 3, further comprising an inducer of apoptosis.
 6. The kit of claim 5, wherein said inducer of apoptosis is a chemotherapeutic agent.
 7. The kit of claim 3, wherein said instructions are for administering said composition to an animal having a hyperproliferative disease.
 8. The kit of claim 7, wherein said hyperproliferative disease is cancer.
 9. A method of preparing a composition consisting essentially of co-crystals of (−)-gossypol with acetic acid in a molar ratio of about 1:1, said method comprising dissolving (−)-gossypol in acetone to form a solution, filtering the solution, adding acetic acid into the solution with mixing until the solution turns turbid, leaving the turbid solution at room temperature then at a reduced temperature of about 0 to 15° C. to form co-crystals, collecting the co-crystals, washing the co-crystals with a solvent, and drying the co-crystals.
 10. The method of claim 9, wherein the reduced temperature is about 4° C.
 11. A pharmaceutical composition comprising co-crystals of (−)-gossypol with acetic acid and a pharmaceutically acceptable carrier, wherein said co-crystals of (−)-gossypol with acetic acid consist essentially of co-crystals of (−)-gossypol with acetic acid.
 12. A pharmaceutical composition comprising co-crystals of (−)-gossypol with acetic acid and a pharmaceutically acceptable carrier, wherein said co-crystals of (−)-gossypol with acetic acid consist essentially of (−) gossypol and acetic acid in a molar ratio of about 1:1.
 13. A method of preparing a pharmaceutical composition comprising combining a composition consisting essentially of co-crystals of (−)-gossypol with acetic acid in a molar ratio of about 1:1 with a pharmaceutically acceptable carrier. 